The culture of hematopoietic cells for transplantation therapies is a rapidly growing area of biotechnology and experimental hematology. As evidenced by recent clinical trials (Brugger et al., New Engl. J. Med., 333, 283-287 (1995); Williams et al., Blood, 87, 1687-1691 (1996); Bertolini et al., Blood, 89, 2679-2688 (1997)), ex vivo expanded hematopoietic cells offer great promise for the reconstitution of in vivo hematopoiesis in patients who have undergone chemotherapy. Other potential applications for ex vivo expansion include production of cycling stem and progenitor cells for gene therapy, expansion of dendritic cells for immunotherapy, and production of red blood cells and platelets for transfusions (McAdams et al., Trends Biotechnol., 14, 388-396 (1996)). Thus, it is likely that the demand for ex vivo expanded hematopoietic cells will increase dramatically.
Hematopoietic cultures are among the most challenging culture systems. The heterogeneous cell population contained in a hematopoietic culture is always changing as a result of the delicate balance between proliferation of certain cell types, their differentiation into other cell types, and the death of various cell populations. The lifespan of cells in culture is likely to depend on cytokine stimulation, as well as on a number of physicochemical parameters, such as pH, dissolved oxygen, and nutrient and metabolite concentrations (McAdams et al., Trends Biotechnol., 14, 341-349 (1996)).
Current enumeration techniques for hematopoietic cultures do not provide real-time analysis of the changing populations. Complete evaluation of the performance of hematopoietic cultures requires the use of assays with long durations, such as the two-week methylcellulose assay to detect progenitor or colony-forming cells (CFC), including colony-forming units-granulocyte/monocyte (CFU-GM) and burst-forming units-erythroid (BFU-E), and the seven-week assay for the very primitive long-term culture-initiating cells (LTC-IC). In this regard, the cell requirements for successful engraftment are often expressed in terms of the number of CFC transplanted (e.g., 2.times.10.sup.5 CFU-GM per kg body weight; Bender et al., J. Hematotherapy, 1, 329-341 (1992)). In contrast to the long assay times, the time period available to determine when to harvest ex-vivo cultures for transplantation therapies is most likely on the order of hours. Currently, only flow cytometry offers this speed of analysis. Flow cytometry can be utilized to quantify cells bearing antigens such as CD34 (primitive progenitors), CD15 and CD11b (granulocyte and monocyte post-progenitors), and gly A (maturing erythrocytes). Even so, sample preparation and measurement, along with data analysis, requires 2-3 hours. Furthermore, when cells bearing the antigen of interest are present at a low concentration, as is often the case for CFC, accurate quantitation may be difficult. Also, it should be noted that, although most CFC present in hematopoietic cell sources (e.g., bone marrow or umbilical cord blood) express the CD34 antigen, CD34 expression by cultured cells is often lost before the CFC content of a culture is depleted. Because of the difficulty in determining when CFU-GM, BFU-E, or other cell populations of interest reach a maximum level, culture endpoints have generally been chosen based on a retrospective analysis of the culture duration that usually yields an acceptable product. While the use of retrospective analysis may be adequate, it is far from optimal due to the heterogeneity in the kinetics of cell expansion (e.g., initial quiescent phase and the time at which various cell populations reach a maximum) for different hematopoietic cell source samples.
Nutrient consumption and by-product accumulation rates are parameters that can be readily measured in real-time. These rates are frequently employed for the control of more traditional cell cultures (for vaccine and protein production), but have been largely overlooked in the evaluation of hematopoietic cultures.
Normal and leukemic human blood cells depend heavily upon glycolysis as their source of energy (Beck, J. Biol. Chem., 232, 251-270 (1958); Beck and Valentine, Cancer Res., 12, 818-822 (1952); Beck and Valentine, Cancer Res., 12, 823-828 (1952)), and the rates of glucose consumption and lactate production can be altered by external stimuli, such as growth factors. Growth factor-stimulated increases in glucose utilization have been demonstrated in cultures of murine macrophages (Hamilton et al., Biochem. Biophys. Res. Commun., 138, 445-454 (1986)) and multipotential hematopoietic cell lines (Whetton et al., EMBO J., 3, 409-413 (1984); Whetton, et al., J. Cell Sci., 84, 93-104 (1986)). Human lymphocytes stimulated to undergo blastogenesis by incubation with phytohemagglutinin (PHA) exhibit increased glucose utilization and lactate production and increased levels of glycolytic pathway enzymes (Hedeskov, Biochem. J., 110, 373-380 (1968); Rogers et al., Ann. Hum. Genet., 43, 213-226 (1980); Kester et al., Arch. Biochem. Biophys., 183, 700-709 (1977)). The findings discussed above for stimulated hematopoietic cells are consistent with those for rapidly dividing cells in general, which are known to exhibit rates of glucose consumption and lactate production that are elevated over those of more slowly growing cells (Hume and Weidemann, J. Natl. Cancer Inst., 62, 3-8 (1979); Newsholme, et al., Biosci. Rep., 5, 393-400 (1985); Lanks and Li, J. Cell. Physiol., 135, 151-155 (1988)). Data regarding oxygen consumption rates in human hematopoietic cultures are scarce, and the published reports have not fully examined the effects of various cell populations on oxygen metabolism. Peng and Palsson (Annals Of Biomedical Engineering, 24, 373-381 (1996)) examined oxygen uptake by human bone marrow cells in modified six-well culture plates. They found that the specific oxygen uptake rate (moles per cell per hour) increased steadily during the first 10 days in culture and then remained steady or increased slightly from days 10-14. Bird et al. (Cancer, 1009-1014 (1951)) measured oxygen uptake by normal human granulocytes.